Purification And Synthesis Of ACE Inhibitory Peptides From Acaudina Molpadioidea Protein Hydrolysate

Establishment of an analysis method for food-derived ACE inhibitory peptides, confirmation of Acaudina molpadioidea protein hydrolytic technics, an antihypertensive effect of hydrolysate by oral administration, purification and structure identification of Acaudina molpadioidea ACE inhibitory peptides and synthesis of ACE inhibitory peptide were studied in this paper.An analysis method of food-derived ACE inhibitory peptides activity in vitro was established in the paper. In the method, the relationship between hippuric acid concentration and peak area exhibited a good linearity in the concentration ranges of 0～200μg/mL and 200～800μg/mL. The analysis method was further validated by captopril, the oyster hydrolysate and the anchovy hydrolysate, and the results indicate that the method is convenient, accurate and suitable for the analysis of food-derived ACE inhibitory peptides activity in vitro.The optimal combined-enzymic hydrolytic conditions of Acaudina molpadioidea protein were confirmed in the paper. The IC50 values of the single enzymic hydrolysates were regarded as evaluative index, and bromelain and alcalase were selected as combined-enzyme. Based on the single enzyme and orthogonal tests, the optimal combined-enzymic hydrolytic conditions of Acaudina molpadioidea protein were confirmed. The combined-enzymic hydrolysate was fractionated into two ranges of molecular weight (﹥2 kDa;﹤2 kDa) using an ultrafiltration membrane bioreactor system. The fraction (﹤2 kDa) brought about a high angiotensin-I-converting enzyme (ACE) inhibitory activity, with IC50 value of 0.62 mg/mL. The fraction (﹤2 kDa) was used as drinks administered to renal hypertensive rats (RHR) for 1 month. The systolic blood pressure and diastolic blood pressure of the RHR were significantly reduced, which indicates an antihypertensive effect by oral administration.Two ACE inhibitory peptides were isolated from the Acaudina molpadioidea combined-enzymic hydrolysate (﹤2 kDa), using the chromatographic methods including gel filtration, ion-exchange chromatography and reversed phase high-performance liquid chromatography. The purified ACE inhibitory peptides were two novel peptides, showing very low similarity to other ACE inhibitory peptide sequences, and were sequenced as MEGAQEAQGD (IC50 value, 15.9μM) and YYLEMDFLLFNY (IC50 value, 30.4μM).The Acaudina molpadioidea ACE inhibitory peptide (MEGAQEAQGD) was synthesized by a method of gradual condensation and fragmental condensation. The purity of the decapeptide was 99.72%, and the yield was as high as 68.2%. The molecular weight and sequence structure of the decapeptide equate with the fact.The characters of the Acaudina molpadioidea ACE inhibitory peptide (MEGAQEAQGD) were studied in the paper. It was found that pre-incubation did not change the inhibitory activity of the peptide, and IC50 values for the peptide before and after pre-incubation with ACE were the same (15.9μM). The ACE inhibitory activity of the peptide showed a change after in vitro incubation with gastrointestinal proteases. It was found that the inhibitory activity of the peptide was intensified by 3 times from IC50 15.9μM to IC50 5.3μM after pepsin digestion. The inhibitory activity of the peptide was increased by a factor of 3.5 from IC50 15.9μM to IC50 4.5μM following further chymotrypsin digestion. Therefore, the peptide from Acaudina molpadioidea can be considered pro-drug type ACE inhibitor.Antihypertensive effect of the decapeptide was evaluated by measuring changes in systolic blood pressure (SBP) of SHR at 1-6 h after oral administration. After 3 h of administrating the ACE inhibitory peptide (3μM/kg per rat), the SBP decreased to 173±2.9 mmHg (compared with negative control group, 192±1.7 mmHg), and the activity was maintained for 5 h. Captopril (3μM/kg per rat) only lowered SBP significantly from 1 to 4 h after administration of the drug. After 4 h of administration, the ACE inhibitory peptide showed more powerfully antihypertensive effect than captopril (P < 0.01).